Serine protease inhibitors or "serpins" are a superfamily of inhibitors involved in the mediation of a variety of biological processes essential to survival of a host. Members of the serpin family play a role in a great number of biological processes including, but not limited to, inflammation, fertilization, tumor migration, neurotropism, and heat shock. Maspin was recently identified and characterized as a protective serpin normally present in mammary epithelium but absent from most mammary carcinoma cell lines. Serpins are found in plants, prokaryotes, insects and animals. Natural mutations and modifications of serpins are correlated with a number of serious disease states. Serpin dysfunction is associated with lung, liver and blood coagulation diseases such as emphysema, liver cirrhosis, thrombosis and pulmonary embolism.
The interaction of serpins with endogenous and microbial proteases produces a spectrum of molecular species, each of which are components of a highly evolutionarily conserved homeostatic mechanism that operates to maintain concentrations of intact, active serpins essential to a host's survival. For example, the serpin-protease complex and the hydrolyzed, inactive form of the intact serpin stimulate the production of interleukin-6, signaling hepatocytes to increase synthesis of the acute phase proteins including a subpopulation of the serpin superfamily of proteins. While serpin-enzyme complexes are rapidly cleared from the circulation, cleaved and intact forms of these complexes can accumulate in local areas of inflammation. This accumulation establishes a complex microenvironment of chemoattractants and inhibitors of chemotaxis as well as activators and inhibitors of neutrophil degranulation, leukotrienes, platelet activating factor (PAF), and superoxide production. The extreme virulence of several pox viruses has been attributed in part to a serpin whose target is cysteine proteinase ICE, the interleukin 1-.beta. converting enzyme.
Through various animal models, it has been demonstrated that uncontrolled serine protease activity is a major mechanism of lung injury and that an appropriate serpin response controls the degree of the injury. For example, antithrombin III (ATIII) in combination with .alpha.-1-protease inhibitor (.alpha.1P1), protected sheep from endotoxin-induced lung injury where the individual serpins were not as effective as the combination. Redens et al., Circ. Shock 1988, 26, 15. Redens et al. also showed that ATIII protects against the development of disseminated intravascular coagulation in endotoxemic rats. Emerson et al., Circ. Shock 1987, 21, 1. A scavenger of H.sub.2 O.sub.2 and a chloromethyl ketone inhibitor of elastase blocked reactive oxygen potentiation of neutrophil elastase-mediated acute edematous lung injury in a rat and .alpha.1P1 diminished bleomycin-mediated pulmonary inflammation as well as subsequent fibrosis. Baird et al., Physiol. 1986, 61, 2224 and Nagai et al., Am. Rev. Resp. Dis. 1992, 145, 651. In another system, however, neutrophil elastase inhibitors, Eglin C and a low molecular weight compound L 658,758, failed to inhibit leukotriene B4-induced-neutrophil-mediated adherence, diapedesis or vascular leakage. Rosengren et al., Am J. Physiol. 1990, 259, H1288. As shown by these studies, inhibitors of proteolytic enzymes administered therapeutically can limit the molecular and cellular mechanism of inflammation and reduce tissue damage.
There are two subfamilies within the serpin superfamily. One family contains proteins for which no cognate serine proteases have yet been identified. Examples of proteins in this subfamily include ovalbumin, angiotensinogen and steroid binding globulins. The second family contains members for which at least one serine protease can be found as an inhibitory target. The subfamily of serpins that inhibit serine proteases have characteristic properties that define the activity of the inhibitor, i.e., second order rate constants for inhibition of their cognate enzyme range between 10.sup.2 and 10.sup.7 M.sup.-1 s.sup.-1 ; the enzyme-inhibitor complex is stable under certain conditions and can be detected as a species with a molecular weight greater than the individual components in SDS polyacrylamide gels; and, a large conformational change occurs upon cleavage of the sessile bond in the reactive center leading to increased thermal stability of the protein. An example of a serpin in this subfamily is .alpha.1-antichymotrypsin (ACT), an inhibitor of chymotrypsin (Chtr). ACT is synthesized predominantly by the liver and is one of the acute phase reactants with levels rising rapidly to more than 5 fold in response to a wide variety of injuries including surgery, acute myocardial infarctions, burns, autoimmune diseases, malignancies, infections and liver allograft rejection. ACT has also been linked with the plasticity of the nervous system and associated with beta amyloid deposits in Alzheimer's disease, in aging brain, Down's syndrome and in the Dutch variant of hereditary cerebral hemorrhage with amyloidosis. It has been demonstrated that both native ACT and recombinant ACT (rACT) inhibit superoxide generation by human neutrophils in suspension.
In general, the reactive loop of an inhibitory serpin is comprised of a relatively short amino acid sequence. The selectivity of setpins for inhibiting a specific type of protease depends on the amino acid sequence of a reactive center region exposed on the surface of the serpin. Jiang H. et al., J. Biol. Chem. 1994, 269, 55-58, demonstrated that the selectivity of serpins from a lepidopteran insect, Manduca sexta, can be altered by changing the amino acid sequence in the reactive loop. It was found that pre-mRNA splicing generates inhibitor diversity and the potential to regulate a variety of proteinases, using the same protein framework joined to different reactive site region cassettes.
Based upon three dimensional structures of complexes formed by small inhibitors (about 50 amino acids) with their target enzymes, it is believed that the P3-P3' region, amino acids 356-361 in ACT, (nomenclature of Schecter I. and Berger A.C. Biochemistry Biophysics Research Communication 1967, 27:157), in the loop of serpins, or so-called "active site region" (or bait region) serves as a primary contact site or binding site with the protease. It has now been found that another part of the loop (from P14 to P9), denoted the "hinge region", is also important for the inhibitory activities of serpins. A method has now been developed for modulating serine protease activity in cells or tissues which comprises selecting a target protease which accumulates in cells or tissues, producing a recombinant serine protease inhibitor having a protease binding site and a hinge region of a reactive center loop which have modified amino acid sequences so that interaction between the inhibitor and the target protease is altered, and contacting cells or tissues with the modified serine protease inhibitor so that serine protease activity is modulated. Compositions capable of effectively modulating serine protease activity which comprise a recombinant serine protease inhibitor having a protease binding site and a hinge region of a reactive loop which have modified amino acid sequences are also provided. These compositions are useful in regulating inflammatory processes. In addition, a method is provided for producing serine protease inhibitors capable of effectively modulating the activity of the serine proteases which comprises determining a sequence of a serine protease inhibitor; identifying a reactive loop of said serine protease inhibitor, said reactive loop containing a first amino acid sequence of a protease binding site and a second amino acid sequence of a hinge region; modifying the first amino acid sequence of said protease binding site so that the selectivity of a recombinant serine protease inhibitor for other proteases is altered; modifying the second amino acid sequence of said hinge region so that said recombinant serine protease inhibitor is capable of effectively modulating the activity of the serine protease; and synthesizing this modified serine protease inhibitor.